TWI568851B - Soybean event pdab9582.814.19.1 detection method - Google Patents

Description

Method for detecting soybean product PDAB9582.814.19.1

The present invention relates to a method for detecting soybean product PDAB9582.814.19.1.

Background of the invention

Genes encoding Cry1F and Cry1Ac synpro (Cry1Ac) are capable of conferring resistance resistance (eg, resistance to lepidopteran insects) to gene transfer plants; and encoding PAT [phosphorothelin acetamidine) The gene of phosphinothricin acetyltransferase can confer tolerance to the herbicide phoshpinothricin [glufosinate] to the gene-transplanting plant. PAT has been successfully demonstrated in soybeans for use as a selectable marker in the production of insect-resistant genetically transformed crops, as well as in the genetically modified crops to give commercial grades to herbicides. Tolerance both.

The expression of foreign genes in plants is known to be affected by their location in the plant genome, perhaps due to the chromatin structure [eg, heterochromatin] or transcriptional regulation elements [For example, an adjacent neighboring integration site of an enhancer] (Weising et al. , Ann. Rev. Genet 22:421-477, 1988). At the same time, the presence of transgenes at different locations in the genome will affect all phenotypes of the plant in different ways. For this reason, in order to identify a feature that is characterized by optimizing the expression of an introduced gene of interest, it is often necessary to screen a number of articles. For example, it has been observed in plants and other organisms that there is a vast variation in the appearance of an introduced gene. There may also be differences in the spatial and temporal maps of expression. For example, differences in the relevant expression of a transgenic gene in various plant tissues may not correspond to those expected from transcriptional regulatory elements present in the introduced gene construct. . For this reason, it is common for commercial purposes to produce hundreds to thousands of different pieces and to screen those pieces for a single piece of the desired gene expression profile and map. A product having the desired level or profile of the transgenic gene expression is useful for the transfer of the transgenic gene into other gene backgrounds by sexual hybridization using conventional breeding methods. The progeny of these crosses maintain the transcriptional gene expression characteristics of the original transformant. This strategy is used to ensure that reliable genes are expressed in many varieties that are well adapted to local growth conditions.

In order to determine whether a sexually progeny progeny contains a transgenic gene or a group of transgenic genes of interest, it is desirable to be able to detect the presence of a particular product. In addition, a method for detecting a particular item would be useful, for example, to comply with pre-market approval and to calibrate food derived from reconstituted crop plants, or for use in environmental monitoring. Monitoring the traits of crops in the field or monitoring the products derived from the harvest of a crop, and the promises of the parties for use in ensuring management or contractual terms.

Any nucleic acid detection method known by the art [including, but not limited to, polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes] It is possible to detect the presence of a genetically modified product. These detection methods generally focus on genetic elements that are frequently used [such as promoters, terminators, markers, etc.] because for many DNA constructs, the coding regions are interchangeable. of. Thus, such methods may not be useful for distinguishing between different articles (especially those produced using the same DNA construct or a very similar construct) unless adjacent DNA of the heterologous DNA inserted is contiguous The DNA sequence is known. For example, a piece-specific PCR analysis for corn product DAS-59122-7 is described in U.S. Patent Application Serial No. 2006/0070139. What is desired is a simple and differentiated method for identifying soybean product pDAB9582.814.19.1.

Summary of invention

The present invention relates to a method for detecting a novel insect-tolerant and herbicide tolerance gene-transformed soybean-transformed article (designated as soybean product pDAB9582.814.19.1). At least 2,500 seeds of a soybean line containing soy product 9582.814.19.1 were deposited as part of this disclosure at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110. The deposit (ATCC Patent Deposit Name PTA-12006) was received by the ATCC on July 21, 2011. For the purposes of the patent procedure, this deposit is made and will be maintained in accordance with and in accordance with the terms of the Budapest Treaty on Seed Hosting.

The DNA of soybean plants containing this product includes characterization The junction/flanking sequences described herein for the position of the inserted DNA within the soybean genome. Sequence identification number: 1 and sequence identification number: 2 for the diagnosis of soybean product pDAB9582.814.19.1. More specifically, the sequence surrounding these junctions (bp 1400/1401 and bp 1536/1537 in sequence identification number: 1 and bp 152/153 in sequence identification number: 2) is used to diagnose soybean product pDAB9582.814.19. .1. An example of a sequence comprising these linkages, which are characteristic of the DNA of soybean containing soybean product pDAB9582.814.19.1, is described in paragraph [0009] below.

The present invention provides a method for detecting a soybean product pDAB9582.814.19.1 in a sample comprising soybean DNA, the method comprising: (a) selectively binding the sample to a sequence identification number: a first primer of at least 10 bp in length of bp 1-1400 or its complement in its complement, and a selectively binding to a bp 1401-1836 of sequence identification number: 1 or a second primer having an insertion sequence of at least 10 bp in length within its complement; and (b) analyzing an amplicon produced between the primers; or selectively binding the sample to To a first primer of at least 10 bp in length of bp 1-152 of sequence identification number: 2 or its complement, and a selective binding to bp 153 of sequence identification number: 2 a second primer of at least 10 bp in length of adjacent sequences within -1550 or its complement; and (c) analyzing an amplicon produced between the primers.

In another embodiment, the present invention provides a method for detecting soybeans The method of pDAB9582.814.19.1, comprising: a) selectively binding the sample to a bp 15-3-1 selected from bp 1-1400 of sequence identification number: 1 and sequence identification number: 2; And a first primer of an adjacent sequence of the group consisting of their complements; and a second primer selectively binding to the sequence identification number: 3 or its complement; b) subjecting the sample to the polymerase chain Reaction; and c) analyzing an amplicon produced between the primers.

In another embodiment, the invention provides an isolated DNA molecule for use in the diagnosis of the soybean product pDAB9582.814.19.1. These molecules include, in addition to sequence identification numbers: 1 and 2, bp 1400-1401 containing sequence identification number: 1 and at least 10 bp of sequence identification number: 1 at least in all directions from bp 1400/1401. 25 bp molecule; 152-153 with sequence ID: 2 and sequence identification number: 2 at least 10 bp of amplicon of at least 25 bp in length from each direction of bp 152/153 junction. Examples are sequence identification number: 1 bp 1385-1415; sequence identification number: 1 bp 1350-1450; sequence identification number: 1 bp 1300-1500; identification number: 1 bp 1200-1600; sequence identification number: 2 Bp 137-168; SEQ ID NO: 2 bp 103-203; and SEQ ID NO: 2 bp 3-303, and their complements.

Furthermore, the present invention provides an analysis for detecting the presence of the target item in a sample (e.g., soy). The analysis can be based on the DNA sequence of the recombinant construct inserted into the soybean genome, as well as the genomic sequence adjacent to the insertion site. Sets and conditions useful in implementing such analysis Also provided.

The present invention relates in part to the selection and analysis of DNA sequences resulting from the insertion of T-DNA from pDAB9582 in the border regions of transgenic soybean lines. These sequences are unique. Based on the insertion and junction sequences, a piece-specific primer can be generated. PCR analysis demonstrated that these products were identified by analyzing the PCR amplicons produced by these product-specific primer pairs. Accordingly, these and other related operational procedures can be used to uniquely identify a soybean line comprising the article of the present invention.

Brief description of the sequence

Sequence Identification Number: 1 is the 5' DNA adjacent border sequence for soybean product 9582.814.19.1. Nucleotides 1-1400 are genomic sequences. Nucleotides 1401-1535 are a rearrangement sequence from pDAB9582. Nucleotides 1536-1836 are insertion sequences.

Sequence Identification Number: 2 is the 3' DNA adjacent border sequence for soybean product 9582.814.19.1. Nucleotides 1-152 are insertion sequences. Nucleotides 1515-1550 are genomic sequences.

Sequence Identification Number: 3 is the DNA sequence of pDAB9582 annotated in Table 1 below.

Sequence identification number: 4 is an oligonucleotide primer 81419_FW3 for confirming the 5' border genomic DNA.

Sequence identification number: 5 is an oligonucleotide primer 81419_RV1 for confirming the 3' border genomic DNA.

Sequence identification number: 6 is an oligonucleotide primer 81419_RV2 for confirming the 3' border genomic DNA.

Sequence identification number: 7 is an oligonucleotide primer 81419_RV3 for confirming the 3' border genomic DNA.

Sequence identification number: 8 is an oligonucleotide primer 5' IREnd-01 for confirming 5' border genomic DNA.

Sequence identification number: 9 is an oligonucleotide primer 5'IREnd-02 for confirming 5' border genomic DNA.

Sequence identification number: 10 is an oligonucleotide primer AtUbi10RV1 for confirming the 5' border genomic DNA.

Sequence identification number: 11 is an oligonucleotide primer AtUbi10RV2 for confirming the 5' border genomic DNA.

Sequence identification number: 12 is an oligonucleotide primer 3'PATEnd05 for confirming 3' border genomic DNA.

Sequence identification number: 13 is an oligonucleotide primer 3'PATEnd06 for confirming the 3' border genomic DNA.

Sequence Identification Number: 14 is the confirmed sequence of the soybean product 9582.814.19.1. These include the 5' genome adjacent sequence, the pDAB9582 T-strand insertion, and the 3' genome adjacent sequence.

Sequence ID: 15 is the oligonucleotide primer 81419_3'F used in the TAQMAN analysis to detect the 3' border of soybean product 9582.814.19.1.

Sequence Identification Number: 16 is the oligonucleotide primer 81419_3'R used in the TAQMAN analysis to detect 3' border soybean product 9582.814.19.1.

Sequence Identification Number: 17 is an oligonucleotide probe 81419_3'P that was used in TAQMAN analysis to detect 3' border soybean product 9582.814.19.1. This probe has a FAM fluorescent moiety added to the 5' end and an MGB quincher added to the 3' end.

Sequence ID: 18 is the oligonucleotide primer GMS116F used in TAQMAN analysis to detect the endogenous reference gene GMFL01-25-J19 (GenBank: AK286292.1).

Sequence ID: 19 is the oligonucleotide primer GMS116R used in TAQMAN analysis to detect the endogenous reference gene GMFL01-25-J19 (GenBank: AK286292.1).

Sequence ID: 20 is the oligonucleotide probe GMS116 used in TAQMAN analysis to detect the endogenous reference gene GMFL01-25-J19 (GenBank: AK286292.1). This probe has a HEX fluorescent moiety added to the 5' end and a BHQ cloning body added to the 3' end.

Simple illustration

Figure 1 is a plastid map of pDAB9582 containing cry1F , cry1Ac and pat .

Figure 2 depicts the position of the primers used to confirm the 5' and 3' boundary sequences of the soybean product pDAB9582.814.19.1.

Figure 3 depicts the genomic sequence alignment of the soybean product pDAB9582.814.19.1.

Figure 4 depicts the primer and probe positions for the TAQMAN analysis of the soybean product pDAB9582.814.19.1.

Detailed description of the preferred embodiment

Both ends of the insert of the product soy product 9582.814.19.1 have been sequenced and characterized. Product specificity analysis was developed. It has also been mapped to chromosome 02 of the soybean genome. The piece can be moved into further elite lines by the gene.

As mentioned slightly above in the background section, the introduction and integration of a transgenic gene into a plant genome involves some random artifacts (hence the name "item" for a particular insertion being expressed). That is, using many transformation techniques [such as Agrobacterium transformation, biolistic transformation (ie, gene gun), and silicon carbide mediated transformation (ie, WHISKERS)], it is not expected that a transgenic gene will become inserted in the genome. Therefore, identification of adjacent plant genomic DNA on both sides of the insert can be important for identifying a plant having a particular insert. For example, a PCR primer can be designed to produce a PCR amplicon across the junction region of the insertion and host genome. This PCR amplicon can be used to identify a unique or different type of insert.

The definitions and examples are provided to aid in the description of the invention and to those skilled in the art to practice the invention. Unless otherwise noted, the terms are to be understood in accordance with the customary use of those skilled in the art. The nomenclature of DNA bases as set forth in 37 CFR §1.822 is used.

As used herein, the term "progeny" means any generation of parental plants comprising the soybean product pDAB9582.814.19.1. Children.

A gene-transferred "product" is transformed into a plant cell by heterologous DNA (i.e., a nucleic acid construct comprising a transgene of interest), which is regenerated to cause insertion of the transgene into the plant. The population of plants within the genome, as well as the selection of a particular plant that is characterized by insertion into a particular genomic location. The term "product" means the original transform comprising the heterologous DNA and the progeny of the transform. The term "product" also means a progeny produced by a sexual outcross between the transformant and another variety comprising the genomic/transgenic DNA. The inserted transgenic DNA and adjacent genomic DNA (genomic/transgenic DNA) from the transformed parent, even after repeated back-crossing to a recurrent parent ) is the presence of the progeny of the cross at the same chromosomal location. The term "product" is also meant to mean a parental line comprising the inserted DNA [eg, the original transformant and the progeny resulting from self-selfing] and a pro that does not contain the inserted DNA. A sexual hybridization of a surrogate line from the insertion of the inserted DNA and immediately adjacent to the inserted DNA that is expected to be transferred to a progeny of the inserted DNA comprising the transgenic gene of interest The original transform of the genomic sequence and the DNA of its progeny.

A "binding sequence" or "boundary sequence" identifies or detects one of the genetic material of a plant across the point at which DNA inserted into the genome is linked to DNA from the natural genome of soybean adjacent to the insertion point. Or other joining sequences are sufficient to diagnose the article. Included are such insertions of the soy products described herein and adjacent DNA of similar length DNA sequence. Specific examples of such diagnostic sequences are provided herein; however, other sequences that partially overlap the insertions or other insertions of the insertions and the genomic sequences are also diagnostic and can be used in accordance with the present invention.

The invention is based in part on the identification of articles using such adjacent, joined and inserted sequences. Related PCR primers and amplicons are included in the present invention. In accordance with the present invention, a PCR assay using an amplicon spanning the inserted DNA and its boundaries can be used to detect or identify commercial gene-transforming soybean varieties or proprietary genes derived from such targets. The line of the soybean line.

These adjacent/joining sequences were used to diagnose the soybean product pDAB9582.814.19.1. Based on these sequences, a feature-specific primer is generated. PCR analysis demonstrated that these soybean lines can be identified in different soybean genotypes by analyzing the PCR amplicons produced by these product-specific primer pairs. Therefore, these and other related operational procedures can be used to uniquely identify these soybean lines. The sequences identified here are unique.

The detection technique of the present invention is particularly useful in joining plant breeding, in which a parent plant containing a piece of interest is crossed to another planting item to try to give one or more additional traits of interest in After the offspring, it is determined which progeny plants contain a particular piece. These PCR assays are beneficial for soybean breeding programs as well as quality control, particularly for commercialized genetically transformed soybean seeds. PCR detection kits for these genetically engineered soybean lines can now also be made and used. This can also be useful for product registration and product management.

Furthermore, adjacent soybean/genomic sequences can be used to specifically identify each inserted genomic location. This information can be used to make molecular marker systems specific to each item. These can be used to speed up breeding strategies and establish linkage data.

Still further, the adjacent sequence information can be used to study and characterize the process of integration of the transgenic genes, genomic integration site characterization, species classification, stability of the transgenic genes and their adjacent sequences, and gene expression {especially Regarding gene silencing, transgenic gene methylation profiles, positional utility, and possible performance-related elements (such as MARS [matrix attachment regions], and the like].

In accordance with all of the present disclosure, it should be apparent that the present invention includes seeds obtainable according to the ATCC registration number as defined in paragraph [0005]. The invention also includes herbicide-tolerant soybean plants grown from a seed deposited with the ATCC deposit number as defined in paragraph [0005]. The invention further includes parts of the plant, such as leaves, tissue samples, seeds produced by the plants, pollen, and the like (wherein they contain cry1F , cry1Ac , pat, and sequence identification numbers: 1 and 2).

As used herein, the term "soybean" means soy ( Glycine max ) and includes all of its varieties that can be bred with a soybean plant.

The DNA molecules of the invention can be used as molecular markers in a marker assisted breeding (MAB) method. DNA molecules of the invention can be used in methods known in the art to identify generally agronomically useful traits (such as AFLP markers, RFLP markers, RAPD markers, SNPs, and SSRs). Used in the middle. Such insect resistance and herbicide-tolerance traits can be followed in a progeny that is crossed with a soybean plant of the invention (or its progeny and any other soybean cultivar or variety) using such MAB methods. Such DNA molecules are markers for this trait, and MAB methods well known in the art can be used to track the herbicide-resistance traits in soybean plants, wherein at least one soybean line of the invention or Their offspring are a parent or ancestor. The method of the invention can be used to identify any soybean variety having the subject matter.

As used herein, a "line" is a group of plants that exhibit little or no genetic variation between individuals for at least one trait. Such lines can be produced by self-pollination and several generations of vegetative propagation from a single parent using tissue or cell culture techniques.

As used herein, the terms "cultivar" and "variety" are synonymous and mean a line that is used in commercial production.

"Stability" or "stable" means that for that particular component, the component is maintained for generations, and preferably at least 3 generations.

Commercial utility is defined as having good plant vigor and high fertility, whereby the crop can be produced by the farmer using customary working equipment, and the oil with the described components can be used routinely. The crushing and extraction equipment is extracted from the seed.

"Agronomically elite" means having an insect resistance and herbicide tolerance in addition to the items resulting from the subject matter. A line of agronomic characteristics (such as yield, maturity, disease resistance, and the like). Any and all of these agronomic features and data points can be used to identify such plants, such as one point or a range of uses, to identify either or both ends of the characteristics of such plants.

As will be appreciated by those skilled in the art in light of this disclosure, a preferred embodiment of a detection kit, for example, can include and/or include a "joining sequence" or a "transfer sequence" (where the soybean genome is adjacent to the insertion sequence) Probes and/or primers for sequence encounters. For example, as indicated in the table below, this includes polynucleotide probes, primers and/or amplicons designed to identify one or both of the junction sequences in which the insertion meets the adjacent sequence. A common design is to have a primer that is heterozygous in the adjacent region, and a primer that is hybridized in the insertion. Each of these primers is often about at least ~15 residues in length. Because of this arrangement, the primers can be used to generate/amplify a detectable amplicon indicative of the presence of a fragment of the invention. These primers can be used to generate an amplicon that spans (and includes) the ligation sequence as indicated above.

The primers that land in the adjacent sequence are typically not designed to be heterozygous more than about 1200 bases or so exceed the junction. Thus, a typical adjacent primer should be designed to contain the beginning of the insertion. At least 15 residues of any one of the 1200 bases of the adjacent sequence. That is, comprising a base pair having a self (or heterozygous) sequence number: 14 from 800 to 1400 and/or Sequence identification number: base pair of 14, a primer of an appropriately sized sequence of 13,897 to 14,497 is within the scope of the invention. Insertion primers can likewise be designed anywhere in the insertion, but the sequence Identification number: 14 base pairs 1400 to 2000 and/or sequence identification number: 14 base pairs 13, 297 to 13, 896 can be used, for example, non-proprietaryly used for this primer design.

One skilled in the art will also recognize that primers and probes can be designed to hybridize under a range of standard heterozygous and/or PCR conditions, wherein the primer or probe is not perfectly complementary to the exemplified sequence. That is, some degree of mismatch can be tolerated. For example, with respect to a primer of about 20 nucleotides, typically one or two or such nucleotides need not bind to the opposite strand if the mismatched base is internal or opposite the amplicon On the end of the primer. A variety of different suitable hybrid conditions are provided below. Synthetic nucleotide analogs [such as inosine] can also be used in the probe. A peptide nucleic acid (PNA) probe as well as DNA and RNA probes can also be used. It is important that such probes and primers are used for diagnostics (capable of uniquely identifying and distinguishing) the presence of a piece of the invention.

It should be noted that errors in PCR amplification can occur, which can result in, for example, a small number of sequencing errors. That is, unless otherwise indicated, the sequences listed herein are determined by generating long amplicons from soybean genomic DNA and then selecting and sequencing the amplicon. It has been found that minor differences and fewer inconsistencies are not uncommon in the sequences generated and determined in this manner, providing amplification for many cycles necessary to generate sufficient amplicons from genomic DNA for sequencing. One skilled in the art will recognize and appreciate that any adjustments required for these types of common sequencing errors or differences are within the scope of the present invention.

It should also be noted that some genomic sequences are to be deleted. It is rare, for example, when a sequence is inserted during the production of a piece of material. Thus, some differences may also occur between, for example, the adjacent sequences of the targets and the genomic sequences listed in GENBANK.

The "inserted" components of the DNA sequence are illustrated in the scheme and are discussed in more detail in the examples below. The DNA polynucleotide sequences of these components or fragments thereof can be used as DNA primers or probes in the methods of the present invention.

In some embodiments of the invention, compositions and methods are provided for detecting the presence of the transgene/genomic insertion region in plants and seeds from a soybean plant and the like. The DNA sequence is provided comprising the 5' transgenic/genomic insertion region junction sequence (between base pairs 800-1400 of sequence identification number: 14) provided herein, the segments thereof, and the like The complement of the illustrated sequences and any of their segments. The DNA sequence is provided comprising the target 3' transgenic/genomic insertion region junction sequence (between base pair 13, 897-14, 497 of sequence identification number: 14), their segments, and such segments as provided herein The complement of the illustrated sequences and any of their segments. The insertion region junction sequence spans the junction between the heterologous DNA inserted into the genome and the DNA from the soybean cell adjacent to the insertion site. These sequences can be used to diagnose this particular item.

Based on these insertion and boundary sequences, a piece-specific primer can be generated. PCR analysis demonstrated that the soybean lines of the present invention can be identified in different soybean genotypes by analyzing the PCR amplicons produced by these product-specific primer pairs. These and other related operating procedures can be used To uniquely identify these soybean lines. Thus, PCR amplicons derived from such primer pairs are unique and can be used to identify these soybean lines.

In some embodiments, a DNA sequence comprising a ligated fragment of the novel transgene/genomic insertion region is an aspect of this invention. Included is a DNA sequence of a sufficient length of polynucleotide comprising a transgenic gene insert and a sufficient length of polynucleotide from one or more of the three soybean genus soybean genome sequences described above and/or Or as a useful sequence for generating a primer sequence for diagnosing one or more of the agglutination products of these soybean plants.

A related embodiment relates to at least 10, 11, 12, 13, 14, 15 comprising a portion of a transgenic gene of a DNA sequence identified herein (such as Sequence ID: 1 and its segments) or its complement. DNA sequences of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more linked nucleotides, and a similar length of adjacent soybean DNA sequences from these sequences or their complements . These sequences are useful as DNA primers in DNA amplification methods. The amplicons produced using these primers are used to diagnose any of the soy products referred to herein. Accordingly, the present invention also encompasses amplicons produced by such DNA primers and homologous primers.

This invention also includes a method of detecting the presence of DNA in a sample (corresponding to the soy product referred to herein). The methods may include: (a) contacting the sample containing the DNA with a pair of primers, when the pair of primers and the DNA from at least one of the soybean articles are used in a nucleic acid amplification reaction, An amplicon for diagnosing the article (a); (b) performing a nucleic acid amplification reaction to thereby produce the amplicon; and (c) detecting the amplicon.

A further detection method of the present invention includes a method of detecting the presence of a DNA in a sample (corresponding to the article), wherein the method comprises: (a) constituting the sample containing the DNA with a severe a probe that hybridizes to DNA from at least one of the soy products under heterozygous conditions and is not hybridized to a control soybean plant (DNA of a non-interested product) under stringent heterozygous conditions (b) subjecting the sample and probe to harsh heterozygous conditions; and (c) detecting the hybridization of the probe to the DNA.

DNA detection kits can be developed using the compositions disclosed herein and methods well known in the art of DNA detection. Such kits are useful for identifying the target soy product DNA in a sample and can be applied to a method for breeding soybean plants containing this DNA. Such kits contain DNA sequences homologous or complementary to, for example, the amplicon disclosed herein, or DNA homologous or complementary to DNA contained in the transgenic gene elements of the target product. sequence. These DNA sequences can be used as probes in DNA amplification reactions or in a DNA hybridization method. The kits may also contain reagents and materials needed to perform the detection method.

A "probe" is a label or reporter molecule attached to a conventionally detectable [such as a radioactive isotope, ligand, chemiluminescent agent or enzyme] Isolated nucleic acid molecule. The probe is complementary to a single strand of a target nucleic acid, as for the present invention, complementary to the genome from one of the soy products (whether from a soybean plant or from a sample comprising DNA from the article) A strand of DNA. The probe according to the invention includes not only Deoxyribonucleic acid or ribonucleic acid and polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of that target DNA sequence.

An "initiator" is a nucleic acid hybrid that is bound to a complementary target DNA strand to form a hybrid between the primer and the target DNA strand, followed by a polymerase (eg, a DNA polymerase) An isolated/synthetic nucleic acid that is elongated along the target DNA strand. The primer pair of the present invention means their use for amplifying a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 in length. , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 , 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 , 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 , 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291, 292 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 polynucleotides or more. These probes and primers are specifically hybridized to a target sequence under highly stringent heterozygous conditions. Preferably, although the ability of the probe to differ from the target sequence and to maintain hybridization to the target sequence can be designed by conventional methods, the probes and primers according to the present invention have a sequence similar to the complete sequence of the target sequence. Sex.

Methods for preparing and using probes and primers are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor. , NY, 1989. A PCR-introduction pair can be derived from a known sequence, for example, by using a computer program intended for that purpose.

Primers and probes according to adjacent DNA and insertion sequences disclosed herein can be used to confirm (and, if necessary, correct) by conventional methods (eg, by re-colonization and sequencing of such sequences). These revealed sequences.

The nucleic acid probes and primers of the invention are hybridized to a target DNA sequence under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a gene-transferred article in a sample. Nucleic acid molecules or fragments thereof can be specifically hybridized to other nucleic acid molecules in some cases. As used herein, two nucleic acid molecules are said to be capable of being specifically heterozygous for each other if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they exhibit complete complementarity. As used herein, a molecule is said to exhibit "complete complementarity" when each nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they are sufficiently heterozygous to allow them to remain bonded to each other under at least the usual "low severity" conditions. Similarly, the molecules are said to be "complementary" if they are sufficiently heterozygous to allow them to remain bonded to each other under the usual "high severity" conditions. The usual harsh conditions are described by Sambrook et al. , 1989. Deviation from complete complementarity is therefore permissible as long as such deviations do not completely exclude the ability of the molecules to form a double-stranded structure. For a nucleic acid molecule to act as a primer or probe, it only needs to be sufficiently complementary in the sequence to be able to form a stable double-stranded structure at the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acid sequence that is specifically hybridized to the complement of the nucleic acid sequence, where it is compared under stringent conditions. The term "stringent conditions" is functionally defined to hybridize a nucleic acid probe to a target nucleic acid by a specific hybrid procedure as discussed in Sambrook et al. , 1989, at 9.52-9.55 (ie, To a particular nucleic acid sequence of interest). See also, Sambrook et al. , 1989 at 9.47-9.52 and 9.56-9.58. Thus, the nucleotide sequences of the invention can be used with respect to their ability to selectively form complementary extended bimolecules with DNA fragments.

Depending on the intended application, we can use heterozygous variation conditions to achieve varying degrees of selectivity of the probe to the target sequence. With regard to applications requiring high selectivity, we will typically employ relatively harsh conditions to form hybrids, for example, we will select relatively low salt and/or high temperature conditions (such as from about 0.02 M to about 0.15 M NaCl is provided at a temperature of from about 50 ° C to about 75 ° C). For example, stringent conditions may involve washing the hybrid membrane at least twice with a highly stringent wash buffer (0.2X SSC, 0.1% SDS, 65 °C). Appropriate harsh conditions to promote DNA heterozygosity [eg, 6.0X Sodium Chloride / Sodium Citrate (SSC) at about 45 ° C, followed by a 2.0X SSC wash at 50 ° C] are those skilled in the art Know. For example, the salt concentration in the cleaning step can be selected from a low severity of about 2.0X SSC at 50 ° C to a high severity of about 0.2X SSC at 50 ° C. In addition, the temperature at the cleaning step can be increased from low severity conditions (at room temperature, about 22 ° C) to high stringency conditions (at about 65 ° C). Both temperature and salt can be varied, or the temperature or salt concentration can be maintained fixed while other variables are altered. These selection conditions tolerate a small mismatch, if any, between the probe and the template or target strand. The detection of DNA sequences by hybridization is well known to those skilled in the art, and the teachings of U.S. Patent Nos. 4,965,188 and 5,176,995 are exemplary of methods of hybrid analysis.

In a particularly preferred embodiment, a nucleic acid of the invention will be The primers (or amplicon or other sequences) (including their complements and fragments), as exemplified or suggested herein, are specifically hybridized under high stringency conditions to one or more of the primers (or amplicon or other sequences thereof) as exemplified or suggested herein. In one aspect of the invention, a labeled nucleic acid molecule of the invention has a nucleic acid sequence as set forth herein as one of the exemplified sequences or complements and/or fragments thereof.

In another aspect of the invention, a labeled nucleic acid molecule of the invention shares between 80% and 100% or between 90% and 100% sequence identity with such nucleic acid sequences. In a further aspect of the invention, a labeled nucleic acid molecule of the invention shares between 95% and 100% sequence identity to the sequence. Such sequences can be used as markers in plant breeding methods to identify progeny of genetic crosses. Hybridization of the probe to the target DNA molecule can be by any number of methods known to those skilled in the art (these can include, but are not limited to, fluorescent labels, radioactive labels, antibody-based labels, and chemistry). Lighted label) is detected.

Regarding the amplification of a target nucleic acid sequence using a particular amplification primer pair (eg, by PCR), "stringent conditions" are conditions that allow the primer pair to hybridize only to the target nucleic acid sequence, one of which has a corresponding The primer of the wild type sequence (or its complement) will bind and preferably produce a unique amplification product (amplicon).

The term "pair (one target sequence) specificity" indicates that a probe or primer is only heterozygous to the target sequence in a sample containing the target sequence under severe heterozygous conditions.

As used herein, "amplified DNA" or "amplicon" means the production of a nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. Things. For example, in order to determine whether the soybean plant resulting from a sexual hybridization contains the genetically-transferred genomic DNA from the soybean plant of the present invention, the DNA extracted from a soybean plant tissue sample may use a primer pair (including a derivative). a nucleic acid amplification method is carried out by introducing an adjacent sequence of the plant's genome adjacent to the insertion site of the inserted heterologous DNA, and a second primer derived from the inserted heterologous DNA) An amplicon is generated for diagnosing the presence of the DNA of the article. The amplicon has a length and has a sequence that is also used to diagnose the article. The amplicon may be of a combined length of one nucleotide base pair in length from the pair of primers; and/or about 2, 3, 4, 5, 6, 7, 8 of the pair of primers 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 , 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 , 159, 160, 161, 162, 163, 164, 1 65, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500, 750, 1000, 1250, 1500, 1750, 2000 or more nucleotide base pairs (plus The range of combined lengths on or minus any of the additions exemplified above. Alternatively, a primer pair can be derived from adjacent sequences on both sides of the inserted DNA, thereby producing an amplicon comprising the entire inserted nucleotide sequence. A plurality of primer pairs derived from the plant genome sequence can be located at a distance from the inserted DNA sequence. This distance can range from about 20,000 nucleotide base pairs to a nucleotide base pair. The use of the term "amplicon" specifically excludes primer dimers that can be formed in a DNA thermal amplification reaction.

Nucleic acid amplification can be accomplished by any of a variety of different nucleic acid amplification methods known to the art, including polymerase chain reaction (PCR). A variety of different amplification methods are known in the art, and are described, inter alia, in U.S. Patent No. 4,683,195 and U.S. Patent No. 4,683,202. PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA. These methods of DNA amplification, as well as other methods known in the art, can be used in the practice of the invention. The sequence of insertion of the heterologous gene into the DNA or from a standard soybean product Adjacent genomic sequences of a fragment can be amplified by the use of primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or the selected DNA. And proved (and corrected, if needed).

The amplicons produced by these methods can be detected by a number of techniques. Agarose gel electrophoresis and staining with ethidium bromide is a well-known method for detecting DNA amplicons. Another such method is Genetic Bit Analysis, in which a DNA oligonucleotide is designed to partially overlap both the adjacent adjacent genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in a well of a microwell. After PCR in the region of interest (using a primer in the inserted sequence and one in the adjacent adjacent genomic sequence), a single strand of PCR product can be hybridized to the immobilized oligonucleotide And as a template, a DNA polymerase and a calibrated ddNTP specific for the next base specificity were used for a single base extension reaction. Reading can be based on fluorescence or ELISA-based. A signal indicates the presence of an insertion/adjacent sequence resulting from successful amplification, hybridization, and single base extension.

Another method is the pyrosequencing technique as described by Winge (Innov. Pharma. Tech. 00: 18-24, 2000). In this method, an oligonucleotide is designed to partially overlap the adjacent genomic DNA and insert DNA. The oligonucleotide is hybridized to a single strand of PCR product from the region of interest (one primer in the inserted sequence and one in the adjacent genomic sequence), and in a DNA polymerase, ATP, It is cultivated in the presence of sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin. DNTPs are added individually and incorporated into a measured optical signal. A light signal indicates the presence of a transgene insertion/adjacent sequence resulting from successful amplification, hybridization, and single or multiple base extension.

Fluorescence polarization is another method that can be used to detect an amplicon of the invention. Following this approach, an oligonucleotide is designed to partially overlap the DNA adjacent to the inserted and inserted DNA. The oligonucleotide is hybridized to a single strand of PCR product from the region of interest (one primer in the inserted DNA and one in the adjacent genomic DNA sequence), and in a DNA polymerase and a fluorescent - Nurtured in the presence of a calibrated ddNTP. Single base extension results in the incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of a transgene insertion/adjacent sequence resulting from successful amplification, hybridization, and single base extension.

TAQMAN (PE Applied Biosystems, Foster City, Calif.) is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe was designed to partially overlap the genomic adjacent and insert DNA junctions. The FRET probe and PCR primer (an primer in the inserted DNA sequence and one in the adjacent genomic sequence) are circulated in the presence of a thermostable polymerase and dNTP. During specific amplification, Taq DNA polymerase clears and releases the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates that the cause is successful Amplification and hybridization of the presence of adjacent/transgenic gene insertion sequences.

Molecular beacons have been described for use in sequence detection. Briefly, a FRET oligonucleotide probe is designed to partially overlap the adjacent genomic and insert DNA junctions. The unique structure of the FRET probe results in a secondary structure that maintains a close proximity to the fluorescent and annihilation moieties. The FRET probe and PCR primer (an primer in the inserted DNA sequence and one in the adjacent genomic sequence) are circulated in the presence of a thermostable polymerase and dNTP. After successful PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and annihilating moieties. A fluorescent signal is generated. A fluorescent signal indicates the presence of a contiguous genomic/transgenic insertion sequence resulting from successful amplification and hybridization.

It has been disclosed that the position of the soybean genome is outstanding for an insertion, and the present invention also encompasses a soybean seed and/or a soybean plant having at least one non-soybean product 9582.814.19.1 inserted in the vicinity of the genomic location. One option is to replace one of the different inserts from one of the pDAB9582.814.19.1 exemplified herein. In these general aspects, the homologous recombination of the target, for example, can be used in accordance with the present invention. This type of technique is the subject of, for example, WO 03/080809 A2 and the corresponding published US application (US 2003/0232410). Thus, the invention encompasses the proximity of all or an identifiable portion comprising a contiguous sequence (SEQ ID NO: 1 bp 1-1400 and SEQ ID NO: 2 bp 153-1550) identified herein. Plant and plant cells that are inserted (instead of or in a multi-copy of the cry1F , cry1Ac or pat genes). An extra copy (or an additional copy) of a cry1F , cry1Ac or pat can also be targeted for insertion by this/these methods.

All of the patents, patent applications, provisional applications, and publications, which are hereby incorporated by reference, are hereby incorporated by reference in their entirety in the extent of the extent of

The following examples are included to illustrate the operating procedures for practicing the invention and to demonstrate certain preferred embodiments of the invention. These examples should not be construed as limiting. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent particular methods used to illustrate preferred modes for its implementation. However, those skilled in the art, in light of this disclosure, will appreciate that many variations can be made in the particular embodiments of the invention, while still obtaining the same or similar results without departing from the spirit and scope of the invention. All percentages are by weight and all solvent mixture ratios are by volume unless otherwise indicated unless otherwise noted.

Unless otherwise indicated, the following abbreviations are used.

Bp base pair

°C Celsius

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetic acid

Kb ki base

Gg microgram

μL microliter

mL ml

M molar mass

PCR polymerase chain reaction

PTU plant transcription unit

SDS sodium dodecyl sulfate

SSC A buffer solution containing a mixture of sodium chloride and sodium citrate, pH 7.0

TBE - a buffer solution containing a mixture of Tris base, boric acid and EDTA, pH 8.3

Specific examples of the invention are further defined in the following examples. It should be understood that these embodiments are provided by way of illustration only. From the above discussion and the embodiments, those skilled in the art can determine the essential features of the invention, and the various changes and modifications of the specific embodiments of the invention can be made without departing from the spirit and scope of the invention. Different uses and conditions. Therefore, various modifications of the specific embodiments of the invention are apparent to those skilled in the <RTIgt; Such modifications are also intended to fall within the scope of the patent application scope attached to the text.

The disclosures of the various references set forth herein are hereby incorporated by reference in its entirety herein in its entirety herein in its entirety herein

Example Example 1: Transformation and selection of Cry1F and Cry1Ac soybean products pDAB9582.814.19.1

Gene-transplanted soybean (soybean) containing the soybean product pDAB9582.814.19.1 was produced via Agrobacterium-mediated transformed tocodyonary node explants. The Agrobacterium tumefaciens strain EHA101 (Hood et al., 1993) carrying the binary vector pDAB9582 ( Fig. 1 ) containing the selectable marker, pat v6 and the gene of interest cry1F v3 and cry1Ac in the T-strand DNA region was Use to start the transformation. The DNA sequence for pDAB9582 is provided in the sequence identification number: 3, which is annotated in Table 1 below.

Agrobacterium-mediated transformation was performed using a modified procedure of Zeng et al. (2004). Briefly, soybean seeds (cv Maverick) germinate on basal medium and cotyledon nodes are isolated and infected with Agrobacterium. Shoot initiation, shoot elongation, and rooting media are supplemented with cefotaxime, timentin, and vancomycin. For the removal of Agrobacterium. The grass selection was used to inhibit the growth of untransformed shoots. The selected shoots are transferred to rooting medium for root development and then transferred to a soil mixture for adaptation to plantlets.

The leaflets of the selected seedlings are smeared with solid grass to screen the putative morphs. The screened seedlings were transferred to a greenhouse, allowed to acclimate and then leaf-coated with solid grass to re-confirm tolerance and are considered to be putative recursions. The screened plants are sampled and used to confirm that the selectable marker gene and/or molecular analysis of the gene of interest is performed. T ₀ plants were allowed to self-pollination in the greenhouse (self fertilize) to create T ₁ seed.

This product (soybean product pDAB9582.814.19.1) was produced from a separate transformed isolate. The T ₁ plant was backcrossed in subsequent generations and moved into the elite variety by the gene. The article was selected based on its unique characteristics [such as a single insertion site, normal Mendelian separation, stability performance, and an excellent combination of potencies (including herbicide tolerance and agronomic performance)]. The following examples contain data used to characterize the soybean product pDAB9582.814.19.1.

Example 2: Characterization of protein expression in soybean product pDAB9582.814.19.1

The biochemical properties of the recombinant Cry1F, Cry1Ac and PAT proteins expressed in soybean product 9582.814.19.1 were characterized. Quantitative enzyme-linked immunosorbent assay (ELISA) is a biochemical property that can be used to characterize these proteins and to confirm the presence of these proteins in soy products. Biochemical analysis known in the art of the performance of 9582.814.19.1.

Example 2.1: Expression of PAT, Cry1F and Cry1Ac proteins in plant tissues

Samples of soybean tissue were isolated from the plants tested and prepared for performance analysis. The PAT protein was extracted from soybean plant tissue with a phosphate buffered physiological saline solution (PBST) containing detergent Tween-20 [containing 0.5% bovine serum albumin (BSA)]. The plant tissue was centrifuged; aqueous supernatant was collected, diluted if necessary with appropriate buffer, and analyzed using a PAT ELISA kit in a sandwich format. This kit was used following the manufacturer's recommended procedures (Envirologix, Portland, ME). This analysis measures the PAT protein being expressed.

The Cry1F protein was extracted from soybean plant tissue in a phosphate buffered physiological saline solution (PBST) containing the detergent Tween-20. The plant tissue was centrifuged; the aqueous supernatant was collected, diluted if necessary with appropriate buffer, and analyzed using a Cry1F ELISA kit in a sandwich format. This kit was used following the manufacturer's recommended protocol (Strategic Diagnostics Inc., Newark, DE). This analysis measures the Cry1F protein that is expressed.

The Cry1Ac protein was extracted from soybean plant tissue with a phosphate buffered physiological saline solution (PBST) containing detergent Tween-20 [containing 0.5% bovine serum albumin (BSA)]. The plant tissue was centrifuged; the aqueous supernatant was collected, diluted if necessary with appropriate buffer, and analyzed using a Cry1Ac ELISA kit in a sandwich format. This kit follows the manufacturer's recommended procedures (Strategic Diagnostics) Inc., Newark, DE) is used. This analysis measures the Cry1Ac protein that is expressed.

Detection analysis was performed to investigate the performance stability and inheritability of the soybean product pDAB9582.814.19.1 vertically (between generations) and horizontally [between lineages within a generation]. Both.

Example 2.2: Expression of Cry1F, Cry1Ac and PAT proteins in plant tissues

The levels of Cry1F, Cry1Ac and PAT proteins were determined in soybean product 9582.814.19.1. The soluble, extractable proteins are measured from soybean leaf tissue using a quantity of enzyme-linked immunosorbent assay (ELISA). From the T ₂ to T ₆ generation soybean product 9582.814.19.1, the performance was stable (no separation) and was consistent across all lineages. Table 2 lists the average performance levels of the gene-transferred proteins in soybean product 9582.814.19.1.

Example 3: Selection and characterization of DNA sequences in the insertion and adjacent border regions of soybean product pDAB9582.814.19.1

To characterize and describe the genomic insertion site, the sequence of the T-DNA border region of the adjacent genome of soybean product pDAB9582.814.19.1 The column is determined. The genomic sequence of the soybean product pDAB9582.814.19.1 was confirmed to contain a 1,400 bp 5' adjacent border sequence (SEQ ID NO: 1) and a 1398 bp 3' adjacent border sequence (SEQ ID NO: 2). PCR amplification according to the soybean sequence pDAB9582.814.19.1 border sequence confirmed that the border regions were unique sequences with soy origin and the joint region was soybean product pDAB9582.814.19.1. These joint areas can be used for the article-specific identification of the soybean product pDAB9582.814.19.1. In addition, the T-strand insertion site is characterized by amplification of a genomic fragment corresponding to the region of the identified adjacent border sequence from the genome of the untransformed soybean. Comparison of the soybean product pDAB9582.814.19.1 with the untransformed genomic sequence revealed an approximately 57 bp deletion from the original locus formed during T-strand integration. Summary, Characterization of this insertion and border sequence of soybean product pDAB9582.814.19.1 indicates that a complete copy of the T-strand from pDAB9582 is present in the soybean genome.

Example 3.1: Confirmation of soybean genome sequence

The 5' and 3' adjacent borders were aligned with a soybean whole genome shotgun sequence from chromosome 02, indicating that the transgenic gene of the soybean product pDAB9582.814.19.1 was inserted on the soybean genome chromosome 02. In order to confirm the insertion site of the soybean product pDAB9582.814.19.1 from the soybean genome, PCR was carried out with different primer pairs ( Fig. 2, Table 3, Table 4 and Table 5 ). Genomic DNA from soybean product pDAB9582.814.19.1 and other gene-transgenic or non-gene-transforming soybean lines was used as a template. To confirm that the 5' border sequences are correct, an primer designed to bind to the At Ubi10 promoter gene element (eg, AtUbi10RV1) and a selected 5' to be bound to the soybean genome chromosome 02 The primer for the end border (the primer designated as 81419_FW3) was used to amplify a DNA segment spanning the 5' end border sequence of the At Ubi10 promoter gene element. Similarly, in order to confirm the selected 3' border sequence, a pat- specific primer (eg 3'PATEnd05) and three primers designed according to the selected 3'-end boundary sequence (named 81419_RV1) , 81419_RV2 and 81419_RV3) were used to amplify DNA segments spanning the pat gene to the 3' border sequence. DNA fragments of the expected size were amplified only from the genomic DNA of soybean product pDAB9582.814.19.1 with individual primer pairs, but no DNA samples from other gene-transforming soybean lines or non-gene transfer controls. The results indicate that the selected 5' and 3' border sequences are adjacent border sequences for the T-strand insertion of the soybean product pDAB9582.814.19.1.

To further confirm this DNA insertion in the soybean genome, a PCR amplification across the soybean border sequence was performed on genomic DNA that did not contain T-strand insertion for soybean product pDAB9582.814.19.1. A primer 81419_FW3 designed according to the 5'-end boundary sequence and a primer 81419-RV3 designed for the 3'-end boundary sequence were used to amplify a DNA segment containing the pDAB9582 T-strand integrated locus. As expected, the completed PCR amplification of the primers of 81419_FW3 and 81419_RV3 yielded approximately 1.5 kb DNA fragment from all other soybean control lines but not pDAB9582.814.19.1. The identified 5' and 3' border sequences of the soybean product pDAB9582.814.19.1 and a soybean whole genome gun sequence from chromosome 02 showed a 57 bp deletion from the original locus. ( Figure 3 ). These results demonstrate that the transgenic gene of the soybean product pDAB8294 is inserted into the site of chromosome 02 of the soybean genome.

Example 4: Characterization of soybean product pDAB9582.814.19.1 via Southern Blot

Southern blot analysis was used to establish an integrated map of the soybean product pDAB9582.814.19.1. These experiments yielded data demonstrating the integration and integrity of these cry1Ac and crylF transgenic genes within the soybean genome. Soybean product pDAB9582.814.19.1 was characterized as a full-length, simple integrated piece containing a single copy of cry1Ac and cry1F PTU from plastid pDAB9582.

Southern blot data suggest that a T-strand fragment was inserted into the genome of the soybean product pDAB9582.814.19.1. Detailed Southern blot analysis using probes specific for the cry1Ac and cry1F genes contained in the T-strand integration region of pDAB9582.814.19.1 and having a cleavage site located within the plastid and generating a pair across The descriptive restriction enzymes of the plastid or the heterozygous fragment inside the plastid and the soybean genomic DNA (boundary fragment) are carried out. Molecular weight indication from heterozygous in the South: The combination of the restriction enzyme and the probe is unique to the article and establishes its identification profile. These analyses also showed that the plastid fragment had been inserted into the soybean genomic DNA without rearranging the cry1Ac and cry1F PTU.

Example 4.1: Soybean leaf sample collection and genomic DNA (gDNA) separation

Genomic DNA was extracted from leaf tissue harvested from individual soybean plants containing the soybean product pDAB9582.814.19.1. In addition, gDNA was isolated from a conventional soybean plant Maverick containing a genetic background representing a substantial strain (lacking the cry1Ac and cry1F genes). Individual genomic DNA was extracted from lyophilized leaf tissue following standard CTAB methods (Sambrook et al (1989)). After extraction, the DNA was spectrally fluorescently quantified using PICO GREEN reagent (Invitrogen, Carlsbad, CA). The DNA was then visualized on an agarose gel to confirm the values from the PICO GREEN analysis and to determine DNA quality.

Example 4.2: DNA digestion and separation

About the southern dot molecularly characterized soybean product pDAB9582.814.19.1, 10 micrograms (10 μg) of genomic DNA was digested. Genomic DNA from soybean pDAB9582.814.19.1 and non-gene-transformed soybean line Maverick was digested by adding approximately 5 units of selected restriction enzyme per μg of DNA and corresponding reaction buffer to each DNA sample. Each sample was incubated overnight at approximately 37 °C. The restriction enzymes AseI , HindIII , NsiI and NdeI (New England Biolabs, Ipswich, MA) were used individually for digestion. The restriction enzymes NotI and ApaLI were used together in a double digestion (New England Biolabs, Ipswich, MA). In addition, a positive hybrid control sample was prepared by combining plastid DNA, pDAB9582, and genomic DNA from the non-gene-transforming soybean variety Maverick. The plastid DNA/genomic DNA cocktail was digested using the same procedures and restriction enzymes as the samples tested.

After the digestions were incubated overnight, 25 μL of QUIK-PRECIP PLUS SOLUTION (Edge Biosystems, Gaithersburg, MD) was added and the digested DNA samples were precipitated with isopropanol. The precipitated DNA pellet was resuspended in 15 μL of 1X loading buffer [0.01% bromophenol blue, 10.0 mM EDTA, 10.0% glycerol, 1.0 mM Tris pH 7.5). The DNA samples and molecular size markers were then electrophoresed on a 0.85% agarose gel in 0.4X TAE buffer (Fisher Scientific, Pittsburgh, PA) at 35 volts for approximately 18-22 hours to achieve fragment separation. The gels were stained with ethidium bromide (Invitrogen, Carlsbad, CA) and the DNA was seen under ultraviolet (UV) light.

Example 4.3: Southern Transfer and Membrane Treatment

Southern blot analysis was performed essentially as described by Memelink et al. (1994). Briefly, after electrophoretic separation and visualization of the DNA fragments, the gels were purified with 0.25 M HCl for approximately 20 minutes and then exposed to a denaturing solution (0.4 M NaOH, 1.5 M NaCl) for approximately 30 minutes. The solution was then neutralized (1.5 M NaCl, 0.5 M Tris pH 7.5) for at least 30 minutes. Southern transfer was performed overnight on a nylon membrane using a wicking system with 10X SSC. After transfer, the DNA was bound to the membrane by UV crosslinking, followed by succinct washing of the membrane with a 2X SSC solution. This method produces a southern ink dot film that is intended for hybridization.

Example 4.4: DNA probe calibration and heterozygous

The DNA fragment bound to the nylon membrane was detected using a calibrated probe ( Table 6 ). The probe was amplified from the plastid pDAB9582 by a PCR-based nucleoside [DIG-11]-dUTP conjugated to digoxigenin (DIG) to a primer using specificity for gene elements. An increased DNA fragment is produced in-house. Generation of DNA probes by PCR synthesis was performed using a PCR DIG PROBE SYNTHESIS KIT (Roche Diagnostics, Indianapolis, IN) following the manufacturer's recommended protocol.

The calibrated probes were analyzed by agarose gel electrophoresis to determine their quality and quantity. A desired number of calibrated probes are then used to hybridize to targets on the nylon membranes using essentially the procedure described for DIG EASY HYB SOLUTION (Roche Diagnostics, Indianapolis, IN). DNA is used to detect such specific fragments. Briefly, nylon membrane dots containing immobilized DNA were succinctly cleaned in 2X SSC and preheated in a hybrid flask at approximately 45-55 ° C in a hybrid flask with 20-25 mL The DIG EASY HYB SOLUTION was pre-mixed for approximately 2 hours. The pre-hybrid solution was then poured out and replaced with ~15 mL of preheated DIG EASY HYB SOLUTION (a specific probe containing a desired amount of denaturation by boiling in a water bath for approximately 5 minutes) ). This hybridization step was then carried out overnight in a hybrid oven at approximately 45-55 °C.

At the end of the probe heterozygos, the DIG EASY HYB SOLUTIONS containing the probes are poured into clean tubes and stored in large -20 ° C. These probes can be reused 2 times according to the manufacturer's recommended operating procedures. The film dots are succinctly rinsed and cleaned in a clean plastic container at room temperature with low severity wash buffer (2X SSC, 0.1% SDS) for approximately 5 minutes, followed by approximately 65 ° C The cells were washed twice with high severity washing buffer (0.1X SSC, 0.1% SDS) for 15 minutes. These film dots were succinctly cleaned with 1X maleic acid buffer from DIG WASH AND BLOCK BUFFER SET (Roche Diagnostics, Indianapolis, IN). About 5 minutes. This was then blocked in 1X blocking buffer for 2 hours, and one with anti-DIG-AP [alkaline phosphatase] antibody (Roche Diagnostics, Indianapolis, IN) in 1X blocking buffer Breeding also lasted for at least 30 minutes. After 2-3 washes with 1X wash buffer, specific DNA probes still bind to these membrane dots, and DIG-calibrated DNA standards use CDP-STAR chemiluminescent nucleic acid detection system (CDP-STAR) CHEMILUMINESCENT NUCLEIC ACID DETECTION SYSTEM) (Roche Diagnostics, Indianapolis, IN) was seen following the manufacturer's recommendations. The dots are exposed to a hemiluminescent film for one or more time points to detect hybrid fragments and to see molecular size standards. The film was developed with an ALL-PRO 100 PLUS film developer (Konica Minolta, Osaka, Japan) and the image was scanned. The number and size of the detected strips are documented for each probe. DIG-LABELED DNA MOLECULAR WEIGHT MARKER II (DIG MWM II) and visible after DIG detection as described DIG-calibrated DNA molecular weight marker VII (DIG MWM VII) was used to determine the size of the hybrid fragment in these southern ink dots.

Example 4.5: Southern blot results

Based on the restriction enzyme sites known to cry1Ac and cry1F PTU, the fragment sizes expected and observed with a particular digestion and probe are provided in Table 7 . Two types of fragments are identified from these digests and hybrids: internal fragments in which the known enzyme address is adjacent to the probe region and are completely contained in the insertion region of the cry1Ac and cry1F PTUs , as well as boundary fragments, one of which A known enzyme site is located at one end of the probe region and a second site is expected in the soybean genome. The size of the boundary segment varies by item, because in most cases, the DNA fragment integration address is unique to each item. The boundary fragments provide a means of locating a restriction enzyme address relative to the integrated DNA and assessing the number of DNA insertions. Southern dot analysis performed on multiple generations of soybeans containing the product pDAB9582.814.19.1 produced a suggestion that a low copy, intact cry1Ac and cry1F PTU from plastid pDAB9582 was inserted into the soybean product pDAB9582.814.19.1 Data within the soybean genome.

The restriction enzymes AseI and NsiI bind to and cleave the unique restriction site of the plastid pDAB9582. These enzymes were then selected to characterize the insertion of the cry1Ac gene in the soybean product pDAB9582.814.19.1. After digestion with AseI and NsiI , respectively, a border fragment of >7286 bp or >9479 bp was expected to be heterozygous for the probe ( Table 7 ). When the AseI and NsiI digestions were used separately, a single cry1Ac hybrid band of approximately 7400 and >10000 bp was observed. Hybridization of the probe to a band of this size implies the presence of an insertion of the single address of the soy genome of the cry1Ac gene in the soybean product pDAB9582.814.19.1. The restriction enzymes NotI and ApaLI were selected to perform a double digestion and release a fragment containing the cry1Ac plant transcription unit (PTU; promoter/gene/terminator) ( Table 7 ). After double digestion with NotI and ApaLI , the expected 4550 bp fragment was observed as a probe. The results obtained by enzymatic digestion of the pDAB9582.814.19.1 samples followed by probe heterozygous indicated that a complete cry1Ac PTU from plastid pDAB9582 was inserted into the soybean genome of soybean product pDAB9582.814.19.1.

These restriction enzymes NdeI and NsiI bind and cleave at the restriction site of plastid pDAB9582. These enzymes were then selected to characterize the insertion of the cry1F gene in the soybean product pDAB9582.814.19.1. After digestion with HindIII , NcoI and NsiI , respectively, border fragments > 5569 bp and > 9479 bp were expected to be heterozygous for the probe ( Table 7 ). When HindIII , NcoI, and NsiI were used, a single cry1F hybrid band of ~7500 bp and >10000 bp was observed. Hybridization of the probe to a band of this size implies the presence of an insertion of the single site of the genome of the cry1F gene in the soybean product pDAB9582.814.19.1. The restriction enzyme HindIII was selected to release a fragment containing the cry1F plant transcription unit (PTU; promoter/gene/terminator) ( Table 7 ). After the HindIII digestion, the predicted 7732 bp fragment was observed as a probe. The result obtained by digesting the soybean product pDAB9582.814.19.1 sample with the enzyme and then the probe hybridization indicated that a complete cry1F PTU from the plastid pDAB952 was inserted into the soybean genome of the soybean product pDAB9582.814.19.1.

Example 4.6: Lack of Skeleton Sequences

Southern blot analysis was also performed to demonstrate the lack of the spectromycin resistance gene ( spec R), the Ori Rep element, and the replication initiation protein trfA (trfA element) in the soybean product pDAB9582.814.19.1. When appropriate positive (pDAB9582 was added to Maverick genomic DNA) and negative (Maverick genomic DNA) controls were included for Southern analysis, no specific heterozygous for either spectinomycin resistance, Ori Rep elements or trfA elements was expected . After NsiI digestion and heterozygous for the specR-specific probe, we expected a 15320 bp size band to be observed in this positive control sample (pDAB9582 was added to Maverick genomic DNA). The specR probe was not hybridized to the sample of the negative control and soybean product pDAB9582.814.19.1. Similarly, after NsiI digestion and heterozygous with the trfA probe, we expected a 15320 bp size band to be detected in the positive control sample (pDAB9582 plus Maverick), but in the negative control and soybean product pDAB9582.814.19 The sample in .1 is lacking. After NdeI digestion and heterozygous with the OriRep-specific probe, another expected size band of 5329 bp was detected in the positive control sample (pDAB9582 was added to Maverick genomic DNA), but in the negative control and soybean Lack of sample pDAB9582.814.19.1. These data indicate that the lack of the spectinomycin resistance gene, the Ori Rep element and the replication initiation protein trfA are in the soybean product pDAB9582.814.19.1.

Example 5: Agronomic and yield field trials and herbicide tolerance

In order to test the agronomic characteristics and efficacy of the soybean product pDAB9582.814.19.1, the article was planted in Puerto Rico in October 2010 and in February 2011 in St. Isabel's efficacy trial. The cultivar Maverick, which was initially transformed to produce the product pDAB9582.814.19.1, was planted at each nursery and included as a control in the experiment. Seeds for T3 nursery are derived from a single plant selection at the T2 stage, and seeds for the T4 nursery are derived from a single plant selection at the T3 stage. The four lineages of the piece were tested for each generation. Each lineage was planted in a plot of 4 columns wide and 7.5 inches long. The interval between the columns is 30 inches. The plot was grown under light for approximately 2.5 weeks to compensate for the short day length in Puerto Rico. Each nursery was sprayed to kill grass (at a ratio of 411 g ae/ha). A plot of control plant Maverick was sprayed with the same proportion of geranium, and a second plot was not sprayed and used as a control comparison for the article.

Data is collected in emergence, general appearance, vigor, height, lodging, and maturity. Herbicide tolerance was assessed by visually viewing chlorosis, leaf necrosis, and plant death ( Table 8 ).

Regarding the comparison of the soybean product pDAB9582.814.19.1 with Maverick, only data from Maverick's unsprayed blocks was used. Regarding the comparison of the treated and unsprayed treatments, the data from the pDAB9582.814.19.1 block that was sprayed with a particular treatment was compared to the data from the Maverick control unsprayed block. Soybean product pDAB9582.814.19.1 showed tolerance to the herbicide herbicide. In contrast, no Maverick plants are tolerant to these herbicide treatments.

Example 6: Characterization of insecticidal activity against soybean product 9582.814.19.1

Field and greenhouse assessments were carried out to characterize Cry1Ac and Cry1F against soybean-trained soybean pests in the soybean product pDAB9582.814.19.1 {including Lithoga genus (Lemon Bean), Soybean californic (Soybean Locust) and grassland The activity of the night moth [fall armyworm]}. The soybean product pDAB9582.814.19.1 was compared to the untransformed soybean variety Maverick to determine the level of plant protection provided by the Cry1F and Cry1 Ac proteins.

The greenhouse test was carried out on plants that were about 4 weeks old. Fifteen plants were used to evaluate the soybean product pDAB9582.814.19.1 and the Maverick control. For each of the insect species tested (Lepidoptera: Moth, Soybean and Spodoptera), three leaf punches were made from each plant for a total of 45 leaf disc/plant/insect species. A 1.4 cm (1.54 cm ² ) leaf die was placed on a test field on top of a 2% water agar that was infected with a newborn larvae and sealed with a porous plastic lid (test arena) ). Mortality and leaf consumption were assessed 4 days after infection (infestation). Larvae that did not respond to the temperature of the probe were considered dead. Leaf damage was assessed by visually scoring the percentage of leaf dies consumed by the insects.

Field evaluations were carried out by collecting leaf samples from the seedlings of the seedlings in Puerto Rico, St. Isabel and sending the leaves to Indianapolis for testing. The nursery community for the soybean product pDAB9582.814.19.1 was planted in February 2011 and consisted of approximately 180 plants arranged in four columns. The columns are 2.3 m long and 76.2 cm apart; individual plants are spaced apart by 5.1 cm in each column. In March 2011, a fully expanded, mainstem trifoliate leaf of approximately 4 nodules under meristem from 10 soybean varieties pDAB9582.814.19.1 plants And 10 'Maverick' plants were excised. The leaves are placed in a calibrated plastic bag (one bag) and sealed. The bagged leaves are packaged and transferred to the laboratory. In the laboratory, one or two 3.33 cm (1.31 in) diameter leaf discs were die molded from the leaves of each three leaf to provide a total of 16 leaf discs. Each leaf disc was placed in a test field on top of a 2% agar that was infected with a newborn Spodoptera litura larvae and sealed with a porous plastic lid. The leaf discs were maintained in a controlled environmental chamber for 7 days, with time mortality and leaf consumption being assessed. No larvae responding to mild exploration It is considered dead. Leaf damage was assessed by visually scoring the percentage of leaf dies consumed by the insects.

The results obtained from these repeated experiments indicated that the soybean product pDAB9582.814.19.1 maintained significantly lower damage to all tested insects than the Maverick control plants. Therefore, the soybean product pDAB9582.814.19.1 has insecticidal activity on this broad host range.

Example 7: Sequence of soybean product pDAB9582.814.19.1

Sequence Identification Number: 14 provides the sequence of the soybean product pDAB9582.814.19.1. This sequence contains a 5' genomic adjacent sequence, a T-strand insertion of pDAB9582, and a 3' genomic adjacent sequence. Regarding sequence identification number: 14, residues 1-1400 are 5' genomic adjacent sequences, residues 1401-1536 are residues from the rearrangement of the pDAB9582 plastid, and 1537-13896 is pDAB9582 T-share The inserted residues, as well as residues 13897-15294, are 3' contiguous sequences. The junction sequence or transition with respect to the 5' end of the insertion thus occurs at residues 1400-1401 of sequence identification number: 14. The junction sequence or transfer with respect to the 3' end of the insert therefore occurs at residues 13896-13897 of sequence identification number: 14.

It should be noted that the progeny from the soybean product pDAB9582.814.19.1 may have a sequence that is slightly detached from the sequence identification number: 14. During the introduction of the soybean product pDAB9582.814.19.1 into the gene transfer and breeding process of plant cells, it is not uncommon for some deletions or other changes to occur for this insertion. Furthermore, errors in PCR amplification can occur, which can result in minor sequencing errors. For example, listed here Adjacent sequences are determined by generating amplicons from soybean genomic DNA and then selecting and sequencing the amplicon. It is not uncommon to find slight differences and minor inconsistencies in the sequences generated and determined in this manner, providing for the amplification of many cycles required to generate sufficient amplicons for sequencing from genomic DNA. Those skilled in the art will recognize and appreciate that any adjustments required for these types of common sequencing errors or inconsistencies are within the scope of the present invention. Thus, the relevant segments of the plastid sequence provided herein may contain some minor variations. Thus, a plant comprising a polynucleotide having some range of identity to the target insert is within the scope of the invention. The identity to the sequence of sequence identification number: 14 may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and one of the sequences illustrated or described herein. A 96%, 99% or 100% sequence identity of a polynucleotide sequence. Thus, some differences between the sequence identification number: 14 and the progeny plants of the soybean product pDAB9582.814.19.1 can be identified and are within the scope of the present invention.

Example 8: Product Specificity TaqMan Analysis

A one-piece specific TAQMAN analysis was developed to detect the presence of the soybean product pDAB9582.814.19.1 and to determine the zygosity status of the plants in the breeding population. The soybean product pDAB9582.814.19.1 contains the T-strand of the binary vector pDAB9582 ( Fig . 1 ). In order to specifically detect the soybean product pDAB9582.814.19.1, the specific TAQMAN primer and probe are inserted in the 5' (sequence identification number: 1) or 3' (sequence identification number: 2) insertion-to-plant bonding The DNA sequence was designed ( Fig . 4 ). A product specificity analysis for the soybean product pDAB9582.814.19.1 was designed using two primers and a target containing the FAM reporter at its 5' end synthesized by Applied Biosystems (ABI) - The specific MGB probe specifically detects a 229 bp DNA fragment spanning the 3' integration junction. This TAQMAN detection method was used for the specificity of the soybean product pDAB9582.814.19.1. It was tested for 7 different products containing Cry1Ac and Cry1F PTU and a soybean specific endogenous reference gene GMFL01-25-J19 (soybean) cDNA, GenBank: AK286292.1) is a dual format control non-gene transgenic soybean variety (Maverick).

Example 8.1: gDNA separation

GDNA samples of 7 different soybean varieties and non-gene transgenic soybean varieties were tested in this study. Genomic DNA was extracted using a modified QIAGEN MAGATTRACT PLANT DNA KIT (Qiagen, Valencia, CA). Eight fresh soybean leaf discs per sample were used for gDNA extraction. For the purposes of this study, the sample was diluted with no DNase water, resulting in a concentration of approximately 10 ng/μL.

Example 8.2: TaqMan analysis and results

The specific TAQMAN primer and probe were designed for a soybean product pDAB9582.814.19.1 specific TAQMAN analysis. These reagents can be used to detect the transgene in the soybean product pDAB9582.814.19.1 under the conditions listed below. Table 9 lists the primer and probe sequences that were specifically developed for the detection of the soybean product pDAB9582.814.19.1.

The multiplex PCR conditions for amplification were as follows: 1X Roche PCR buffer, 0.4 μM product specific forward primer, 0.4 μM product specific reverse primer, 0.4 μM primer GMS116 F, 0.4 μM Primer GMS116 R, 0.2 μM fragment specific probe, 0.2 μM GMS116 probe, 0.1% PVP, 6-20 ng gDNA in a total reaction of 10 μL. The cocktail was augmented using the following conditions: i) 95 ° C for 10 minutes, ii) 95 ° C for 10 seconds, iii) 60 ° C for 40 seconds, iv) repeating steps ii-iii for 40 cycles, v) maintaining 40 ° C . Real-time PCR was performed on ROCHE LIGHTCYCLER 480. The data analysis was based on measuring the crossing point (Cp value) determined by the LIGHTCYCLER 480 software (which is the number of PCR cycles at which the ratio of changes in fluorescence reached its maximum).

The TAQMAN detection method for soybean product pDAB9582.814.19.1 was tested for 7 different products containing Cry1Ac and Cry1F PTU and a soybean specific endogenous reference gene GMFL01-25-J19 (GenBank: AK286292.1 A dual-format non-gene-transforming soybean variety. The assay specifically detects the soybean product pDAB9582.814.19.1 and does not produce or amplify any of the controls from the controls (ie, those containing Cry1Ac and Cry1F PTU and a non-gene transgenic soybean variety) The false positive result. These article-specific primers and probes can be used to detect the soybean product pDAB9582.814.19.1, and these conditions and reagents can be applied to the zygosity analysis.

The principles of the present invention have been illustrated and described, it will be apparent to those skilled in the art that the invention can be We request all modifications in the spirit and scope of the patent application scope attached to the text.

All of the publications and published patent documents cited in this specification are hereby incorporated by reference in their entirety as if the individual disclosures or patent applications are expressly or individually The same degree of reference material.

Figure 1 is a plastid map of pDAB9582 containing cry1F , cry1Ac and pat .

Figure 2 depicts the position of the primers used to confirm the 5' and 3' boundary sequences of the soybean product pDAB9582.814.19.1.

Figure 3 depicts the genomic sequence alignment of the soybean product pDAB9582.814.19.1.

Figure 4 depicts the primer and probe positions for the TAQMAN analysis of the soybean product pDAB9582.814.19.1.

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Claims (1)

A method for detecting soybean product pDAB9582.814.19.1 in a sample containing soybean DNA, the method comprising: (a) selectively binding the sample to a sequence identification number: 1 bp 1 a first primer of -1400 or a flanking sequence within its complement that is at least 10 bp in length, and an insert that selectively binds to a bp 1401-1836 of a sequence identification number: 1 or its complement a second primer contact of at least 10 bp in length; and analyzing an amplicon generated between the primers; or (b) selectively binding the sample to a sequence identification number: 2 a first primer of 1-152 or its complement insertion sequence of at least 10 bp in length, and a flanking sequence selectively binding to bp 153-1550 of sequence identification number: 2 or its complement A second primer of at least 10 bp in length is contacted; and an amplicon generated between the primers is analyzed.